This disclosure relates to the microfluidic selection of library elements.
It is desirable in virtually every area of the biomedical sciences to have systems that are based on chemical or biochemical assays for determining the presence and quantity of particular analytes. This desire ranges from the basic science research lab, where biochemical pathways are being mapped out and their functions correlated to disease processes, to clinical diagnostics, where patients are routinely monitored for levels of clinically relevant analytes. Other areas include pharmaceutical research and drug discovery applications, DNA testing, veterinary, food, and environmental applications. In all of these cases, the presence and quantity of a specific analyte or group of analytes, has to be determined.
For analysis in the fields of pharmacology, genetics, chemistry, biochemistry, biotechnology, molecular biology and others, it is often useful to detect the presence of one or more molecular structures and characterize interactions between molecular structures. The molecular structures of interest generally include antibodies, antigens, metabolites, proteins, drugs, small molecules, enzymes, nucleic acids, and other ligands and analytes. The molecular structures can also be inside or outside cells and microorganisms. In medicine, for example, it is very useful to determine the existence of cellular constituents such as receptors or cytokines, or antibodies and antigens which serve as markers for various disease processes, which exist naturally in physiological fluids or which have been introduced into the system. In genetic analyses, fragment DNA and RNA sequence analysis are very useful in diagnostics, genetic testing and research, agriculture, and pharmaceutical development. Because of the rapidly advancing state of molecular cell biology and understanding of normal and diseased systems, there always exists an increasing need for newer, more rapid, and more accurate methods of detection.
A useful technique for the identification of such molecular structures as well as interactions between molecular structures is high throughput screening of large collections of chemicals or biochemicals, often referred to as “libraries”. Most high-throughput screens measure the action of compounds on a single molecular phenomenon, e.g., a particular enzymatic activity that is thought to play a role in some physiological system such as a disease state. Prior to the screening process, the elements of such libraries have not been demonstrated to have action on the molecular phenomenon measured by the screen or the disease state in which the molecular phenomena plays a role. Such a screen is designed to identify compounds that affect that particular molecular phenomenon, so that the physiological system in which the phenomenon plays a role may be impinged upon with the identified compounds.
Screening of libraries is often conducted by using microtiter plates and bead based screening. In screening a library using a microtiter plate, a microtiter plate well is coated with a target of interest (e.g., a receptor). Bacteriophage libraries, more commonly called phage libraries, are often used for screening purposes. In these libraries, chemical variability is introduced in the genome of the phages and because a large number of phages can be contained in a small volume of library, large chemical diversity in the phages can be achieved. In the phage libraries, the variable part of the genome of a phage can be expressed and displayed as a coat protein. Therefore, screening a phage library can be accomplished by looking for interactions between a receptor of interest and a particular protein displayed on the surface of the phage. A phage library is then placed in contact with a well of an analytical device that contains a receptor of interest. Some of the phages bind to the receptor. The well is then washed to remove those phages that are not bound to the receptor. After removal of the unbound phages, those phages that are bound to the receptor are eluted. The DNA of some of the bound phages is then sequenced to assess the quality of the screening. The eluted phages are then copied to increase their numbers (amplification). The foregoing steps are then repeated until the genetic sequences of the bound phages show “consensus”. The emergence of a consensus shows that screening has resulted in extracting from the library one or a few phages that are able to bind the receptor with equal probability.
In bead based screening, a bead of latex, silica, or other suitable material having an average particle size of about 1 to about 10 micrometers is coated with a receptor of interest. The phage library is allowed to interact with the beads freely in solution. Unbound phages and beads are separated using either centrifugation or particle sorting machines based on multiple technologies (magnetic bead, dielectrophoresis, fluorescence). Phages bound to the bead are eluted. As noted above, the eluted phages are subjected to amplification followed by the same series of steps described above to show consensus.
Because of the number of steps, both of the aforementioned methods involving microtiter plates and bead based screening are expensive, time consuming and labor intensive. For example, a phage library can cost around $1,000 to purchase and 2 to 4 rounds of screening generally take about 3 weeks. In addition, both of the above methods use multiple cycles, which opens the method to contamination as well as degradation in the quality of results.
The screening and identification of multiple elements from a library is even more difficult. While multiple elements can be screened simultaneously, information pertaining to the specificity of the interaction of the elements with each target is not easily obtained. For example, screening a library to find logical binders, i.e., a binder that binds to a first target and a second target, a first target or a second target, or a first target but not a second target is very difficult and compounds the complexity of the screening work. In other words, screening a library to find a binder for the first target and the second target uses more than twice the work of screening against the first target followed by screening against the second target.
It is therefore desirable to have a method that can be used for screening phage libraries efficiently and inexpensively.